MOLECULAR BASIS OF IMMUNOGLOBULIN GENE HYPERMUTATION

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Further diversification of the preimmune antibody repertoire is provided by a unique process of immunoglobulin (Ig) gene somatic mutation. This process is tightly focused to rearranged VDJ segments and immediate flanking sequences. The mutation rate approaches 10-3/b.p./generation which is four orders of magnitude greater than that estimated for other eukaryotic genes. This process may be responsible for the development of self reactive antibody clones in autoimmune disorders and has also been implicated in the activation of certain oncogenes in B cell tumors. The biologic bases of Ig gene somatic hypermutation is unknown. We propose to localize regions of the Ig locus responsible for this process by cloning portions of the gene in close proximity to a marker gene, sup F. Mutations initiated and focused by Ig fragments may be expected to be introduced into this flanking target gene similar to what occurs in naturally occurring VDJ joinings and in non Ig sequences at the site of chromosomal translocations. Such changes will be recognized by a sensitive microbiological assay when this plasmid is used to transform indicator strains of E. Coli. A series of vector systems will be used to cycle Ig segments through various lymphoid environments ranging from cell lines to transgenic animals. Comparison of mutation rates initiated by different V regions may allow detection of recognition sequences highly sensitive to this process. Comparison of mutations that occur in different cell lineages may identify the presence of developmentally regulated cellular components which influence the mutational process. In addition to identifying the cellular and molecular basis of Ig Hypermutation, these experiments will determine the impact of this process on antibody diversity in the absence of antigen selection. These studies will lead to a broader understanding of the role of Ig mutation in the normal immune response, its potential contribution to autoimmunity and B cell malignancy, and will provide important insights into more general aspects of DNA mutagenesis.